MASP-2, a complement-fixing enzyme, and uses for it

ABSTRACT

The invention relates to the discovery and characterization of mannan binding lectin-associated serine protease-2 (MASP-2), a new serine protease that acts in the MBLectin complement fixation pathway.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit from Provisional ApplicationSerial No. 60/042,678, filed Apr. 2, 1997, hereby incorporated byreference.

FIELD OF THE INVENTION

[0002] The invention is in the general field of innate pathways forcomplement fixation involving mannan-binding lectin (MBL), also termedmannan binding protein.

BACKGROUND OF THE INVENTION

[0003] The complement system comprises a complex array of enzymes andnon-enzymatic proteins of importance to the function of the innate aswell as the adaptive immune defense¹. Until recently two modes ofactivation were known, the classical pathway initiated byantibody-antigen complexes and the alternative pathway initiated bycertain structures on microbial surfaces. A third, novelantibody-independent pathway of complement activation has beendescribed². This pathway is initiated when mannan-binding lectin (MBL,first described as mannan-binding protein, MBP, see Ezekowitz, U.S. Pat.No. 5,270,199) binds to carbohydrates.

[0004] MBL is structurally related to the C1q subcomponent of componentC1 of complement, and it appears that MBL activates the complementsystem via an associated serine protease termed MASP⁴ or p100⁵, which issimilar to the C1r and C1s components of the classical pathway. The newcomplement activation pathway is called the MBLectin pathway. Accordingto the mechanism postulated for this pathway, MBL binds to specificcarbohydrate structures found on the surface of a range ofmicroorganisms including bacteria, yeast, parasitic protozoa andviruses⁶, and its antimicrobial activity results from activation of theterminal, lytic complement pathway components⁷ or promotingphagocytosis⁸.

[0005] Reportedly, the level of MBL in plasma may be geneticallydetermined^(9,10,11). MBL deficiency is associated with susceptibilityto frequent infections with a variety of microorganisms inchildhood^(12,13), and, possibly, in adults^(13,14). Recent informationassociates MBL deficiency with HIV infection and with more rapid deathfollowing development of AIDS^(15,16). MBL binds to the a galactosylform of IgG (G0), which is found at elevated concentrations inrheumatoid arthritis patients, and then activates complement¹⁷. MBLdeficiency is also associated with a predisposition to recurrentspontaneous abortions¹⁸, and also to development of systemic lupuserythrematosus¹⁹.

[0006] In the first clinical reconstitution trial, an infantMBL-deficient girl suffering from recurrent infections was apparentlycured by injections with purified MBL²⁰. For a recent review on MBL, seeref. 6.

[0007] Relatively high frequencies of MBL mutations associated withMBL-deficiency have been reported in all populations studied. Thisobservation has led to the hypothesis that MBL may, in certain cases,render the individual more susceptible to certain intracellularinfectious agents exploiting MBL to gain access to the target tissues²¹.Since MBL is a very powerful activator of the complement system, it mayalso be that inexpedient activation through microbial carbohydrates orendotoxins can lead to damaging inflammatory responses¹⁰. Thus, theoverall survival of a population may benefit from the wide individualrange of MBL concentrations.

[0008] MASP-1 (MBP-associated serine protease, MASP) is a serineprotease similar in structure to C1r and C1s of the complement pathwayalthough it has a histidine loop structure of the type found in trypsinand trypsin-like serine proteases. MASP-1 has been found to be involvedin complement activation by MBL. A cDNA clone encoding MASP-1 has beenreported that encodes a putative leader peptide of 19 amino acidsfollowed by 680 amino acid residues predicted to form the maturepeptide.

[0009] An abstract reports the existence of a second MASP, termedMASP-2.²².

SUMMARY OF THE INVENTION

[0010] The invention relates to the isolation and characterization of amannan-binding lectin (MBL) associated serine protease (MASP-2). MASP-2shows some homology with the previously reported MASP (MASP-1) and thetwo C1q-associated serine proteases, C1r and C1s. MBL alone does notprovide a functional MBLectin pathway of complement activation.

[0011] We have cloned and sequenced the cDNA encoding MASP-2. Inaddition, we have produced anti-MASP-2 antibody and constructed an assayfor the estimation of MASP-2 in body fluids or tissue extracts.Furthermore, we have constructed quantitative assays for thedetermination of MASP-2 activity in serum or plasma, either when presentas part of the MBL/MASP complex or as free MASP not associated with MBL.

[0012] Thus, one aspect of the invention features substantially puremannin-binding lectin associated serine protease-2 (MASP-2) polypeptidesand nucleic acids encoding such polypeptides. Preferably, the MASP-2polypeptide retains one or more MASP-2 functions, such as being capableof associating with mannan-binding lectin (MBL), serine proteaseactivity, or the MASP-2 activity in an in vitro assay for MBLectincomplement pathway function, e.g., in one of the assay systems describedbelow. Some MASP-2 polypeptides according to the invention, e.g., thoseused in binding assays, may be conjugated to a label so as to permitdetection and/or quantification of their presence in the assay. Suitablelabels include enzymes which generate a signal (e.g., visibleabsorption), fluorophores, radionuclides, etc. Other MASP-2 polypeptidesare capable of competitively inhibiting one of the MASP-2 activitiesdescribed above, and thereby are useful in evaluating MASP-2 function.Other MASP-2 polypeptides are useful antigens or haptens for producingantibodies as described below. Compounds which competitively inhibit aMASP-2 activity are also featured. Preferably, such compounds act byinhibiting the serine protease activity of MASP-2 or of a fragment ofMASP-2. Such compounds may include fragments of MBL or of MASP-2 whichcompetitively inhibit the MBL-MASP-2 interactions critical to complementactivation by the MBLectin pathway, as well as compounds, e.g., peptidefragments, which inhibit the catalytic cleavage of complement factors C4and C2 by MASP-2.

[0013] Specific polypeptides according to this aspect of the inventioninclude: a) a polypeptide with a molecular mass of 20K and containingthe sequence identified as SEQ ID NO:1 [T P L G P K W P E P V F G R L AS P G F P G E Y A N D Q E R R W T L T A P P G Y R]; b) a polypeptidewith a molecular mass of 52K and containing the sequence identified asSEQ ID NO:1; c) a polypeptide having the complete amino acid sequence ofFIG. 6 (SEQ ID NO:2).

[0014] Another aspect of the invention includes an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptide havingsequence that is at least 85% identical to the sequence of SEQ ID NO:2.

[0015] The invention also features isolated nucleic acid sequencesencoding the above mannan-binding lectin associated serine protease-2(MASP-2) polypeptides. Such nucleic acid sequences may be included innucleic acid vectors (e.g., expression vectors including those withregulatory nucleic acid elements permitting expression of recombinantnucleic acid in an expression system).

[0016] The invention also features antibodies that selectively bind toMASP-2. Such antibodies may be made by any of the well known techniquesincluding polyclonal and monoclonal antibody techniques. The antibodymay be coupled to a compound comprising a detectable marker, so that itcan be used, e.g. in an assay to detect MASP-2.

[0017] The polypeptides or antibodies may be formulated intopharmaceutical compositions and administered as therapeutics asdescribed below.

[0018] The invention also features methods for detecting mannan-bindinglectin associated serine protease-2 (MASP-2). The method comprises;obtaining a biological sample, contacting the biological sample with aMASP-2 polypeptide specific binding partner, and detecting the boundcomplexes, if any, as an indication of the presence of MASP-2 in thebiological sample. The binding partner used in the assay may be anantibody, or the assay for MASP-2 may test for complement fixingactivity. These assays for MASP-2 may also be used for quantitativeassays of MASP-2 or MASP-2 activity in biological samples. One of thebinding parners may be specific for MBK thus allowing for the detectionof MBL/MASP-2 complexes.

[0019] Methods for detecting MASP-2 nucleic acid expression are includedin the invention. These methods comprise detecting RNA having a sequenceencoding a MASP-2 polypeptide by mixing the sample with a nucleic acidprobe that specifically hybridizes under stringent conditions to anucleic acid sequence encoding all or a fragment of MASP-2.

[0020] The invention also features methods for treating patientsdeficient in MASP-2 or MASP-2 activity. This is accomplished byadministering to the patient MASP-2 polypeptide or nucleic acid encodingMASP-2. Because it is sometimes desirable to inhibit MASP-2 activity,the invention includes a method for inhibiting the activity of MASP-2 byadministering to the patient a compound that inhibits expression oractivity of MASP-2. Inhibition of MASP-2 activity may also be achievedby administering a MASP-2 anti-sense nucleic acid sequence.

[0021] The invention features an assay for polymorphisms in the nucleicacid sequence encoding MASP-2. A method of detecting the presence ofMASP-2-encoding nucleic acid in a sample is claimed. As an example, themethod may include mixing the sample with at least one nucleic acidprobe capable of forming a complex with MASP-2-encoding nucleic acidunder stringent conditions, and determining whether the probe is boundto sample nucleic acid. The invention thus includes nucleic acid probecapable of forming a complex with MASP-2-encoding nucleic acid understringent conditions.

[0022] The invention features an assay for polymorphisms in thepolypeptide sequence comprising MASP-2 or its precursor.

[0023] MASP-2 assays are useful for the determination of MASP-2 levelsand MASP-2 activity in patients suffering from various diseases such asinfections, inflammatory diseases and spontaneous recurrent abortion.MASP-2 is useful for the treatment of infections when MASP-2 function issuboptimal, and inhibition of MASP-2 activity is useful for regulationof inflammation and adverse effects caused by activity of the MBLectinpathway.

[0024] By “mannan-binding lectin associated serine protease-2” or“MASP-2” is meant the polypeptide or activity called “mannan-bindingprotein associated serine protease-2” or “mannose-binding proteinassociated serine protease” or any other polypeptide having substantialsequence identity with SEQ ID NO:2.

[0025] The terms “protein” and “polypeptide” are used herein to describeany chain of amino acids, regardless of length or post-translationalmodification (for example, glycosylation or phosphorylation). Thus, theterm “MASP-2 polypeptide” includes full-length, naturally occurringMASP-2 protein, as well as recombinantly or synthetically producedpolypeptide that corresponds to a full-length naturally occurring MASP-2polypeptide, or to particular domains or portions of a naturallyoccurring protein. The term also encompassses mature MASP-2 which has anadded amino-terminal methionine (which is useful for expression inprokaryotic cells).

[0026] The term “purified” as used herein refers to a nucleic acid orpeptide that is substantially free of cellular material, viral material,or culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.

[0027] By “isolated nucleic acid molecule” is meant a nucleic acidmolecule that is separated in any way from sequences in the naturallyoccurring genome of an organism. Thus, the term “isolated nucleic acidmolecule” includes nucleic acid molecules which are not naturallyoccurring, e.g., nucleic acid molecules created by recombinant DNAtechniques.

[0028] The term “nucleic acid molecule” encompasses both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. Where single-stranded, the nucleic acid may be a sensestrand or an antisense strand.

[0029] The invention also encompasses nucleic acid molecules thathybridize, preferably under stringent conditions, to a nucleic acidmolecule encoding an MASP-2 polypeptide (e.g., a nucleic-acid moleculehaving the sequence encoding SEQ ID NO:2, e.g., the protein encodingportion of the cDNA sequence shown in FIG. 6—SEQ ID NO:3). In addition,the invention encompasses nucleic acid molecules that hybridize,preferably under stringent conditions, to a nucleic acid molecule havingthe sequence of the MASP-2 encoding cDNA contained in a clone.Preferably the hybridizing nucleic acid molecule consists of 400, morepreferably 200 nucleotides.

[0030] Preferred hybridizing nucleic acid molecules encode an activitypossessed by MASP-2, e.g., they bind MBL and have activity in theMBLectin complement pathway, and can act as serine proteases.

[0031] The invention also features substantially pure or isolated MASP-2polypeptides, preferably those that correspond to various functionaldomains of MASP-2, or fragments thereof. The polypeptides of theinvention encompass amino acid sequences that are substantiallyidentical to the amino acid sequence shown in FIG. 6.

[0032] The polypeptides of the invention can also be chemicallysynthesized, synthesized by recombinant technology, or they can bepurified from tissues in which they are naturally expressed, accordingto standard biochemical methods of purification.

[0033] Also included in the invention are “functional polypeptides”which possess one or more of the biological functions or activities ofMASP-2. These functions or activities are described in detail in thespecification. A functional polypeptide is also considered within thescope of the invention if it serves as an antigen for production ofantibodies that specifically bind to MASP-2 or fragments (particularlydeterminant containing fragments) thereof.

[0034] The functional polypeptides may contain a primary amino acidsequence that has been modified from those disclosed herein. Preferablythese modifications consist of conservative amino acid substitutions, asdescribed herein. The polypeptides may be substituted in any mannerdesigned to promote or delay their catabolism (increase theirhalf-life).

[0035] Polypeptides or other compounds of interest are said to be“substantially pure” when they are distinct from any naturally occuringcomposition, and suitable for at least one of the uses proposed herein.While preparations that are only slightly altered with respect tonaturally occuring substances may be somewhat useful, more typically,the preparations are at least 10% by weight (dry weight) the compound ofinterest. Preferably, the preparation is at least 60%, more preferablyat least 75%, and most preferably at least 90%, by weight the compoundof interest. Purity can be measured by any appropriate standard method,for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

[0036] A polypeptide or nucleic acid molecule is “substantiallyidentical” to a reference polypeptide or nucleic acid molecule if it hasa sequence that is at least 85%, preferably at least 90%, and morepreferably at least 95%, 98%, or 99% identical to the sequence of thereference polypeptide or nucleic acid molecule.

[0037] Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length. Of course, many other polypeptideswill meet the same criteria.

[0038] In the case of polypeptide sequences which are less than 100%identical to a reference sequence, the non-identical positions arepreferably, but not necessarily, conservative substitutions for thereference sequence. Conservative substitutions typically includesubstitutions within the following groups: glycine and alanine; valine,isoleucine, and leucine; aspartic acid and glutamic acid; asparagine andglutamine; serine and threonine; lysine and arginine; and phenylalanineand tyrosine.

[0039] For polypeptides, the length of the reference polypeptidesequence will generally be at least 16 amino acids, preferably at least20 amino acids, more preferably at least 25 amino acids, and mostpreferably 35 amino acids, 50 amino acids, or 100 amino acids. Fornucleic acids, the length of the reference nucleic acid sequence willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 100 nucleotides or 300 nucleotides.

[0040] Sequence identity can be measured using sequence analysissoftware (for example, the Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705), with the defaultparameters as specified therein.

[0041] The nucleic acid molecules of the invention can be inserted intoa vector, as described below, which will facilitate expression of theinsert. The nucleic acid molecules and the polypeptides they encode canbe used directly as diagnostic or therapeutic agents, or can be used(directly in the case of the polypeptide or indirectly in the case of anucleic acid molecule) to generate antibodies that, in turn, areclinically useful as a therapeutic or diagnostic agent. Accordingly,vectors containing the nucleic acid of the invention, cells transfectedwith these vectors, the polypeptides expressed, and antibodiesgenerated, against either the entire polypeptide or an antigenicfragment thereof, are among the preferred embodiments.

[0042] The invention also features antibodies, e.g., monoclonal,polyclonal, and engineered antibodies, which specifically bind MASP-2.By “specifically binds” is meant an antibody that recognizes and bindsto a particular antigen, e.g., the MASP-2 polypeptide of the invention,but which does not substantially recognize or bind to other molecules ina sample, e.g., a biological sample, which includes MASP-2. Referencesto constructs of antibody (or fragment thereof) coupled to a compoundcomprising a detectable marker includes constructs made by anytechnique, including chemical means or by recombinant techniques.

[0043] The invention also features antagonists and agonists of MASP-2that can inhibit or enhance one or more of the functions or activitiesof MASP-2, respectively. Suitable antagonists can include smallmolecules (i.e., molecules with a molecular weight below about 500),large molecules (i.e., molecules with a molecular weight above about500), antibodies that bind and “neutralize” MASP-2 (as described below),polypeptides which compete with a native form of MASP-2 for binding to aprotein, e.g., MBL, and nucleic acid molecules that interfere withtranscription of MASP-2 (for example, antisense nucleic acid moleculesand ribozymes). Agonists of MASP-2 also include small and largemolecules, and antibodies other than “neutralizing” antibodies.

[0044] The invention also features molecules which can increase ordecrease the expression of MASP-2 (e.g., by influencing transcription ortranslation). Small molecules (i.e., molecules with a molecular weightbelow about 500), large molecules (i.e., molecules with a molecularweight above about 500), and nucleic acid molecules that can be used toinhibit the expression of MASP-2 (for example, antisense and ribozymemolecules) or to enhance their expression (for example, expressionconstructs that place nucleic acid sequences encoding MASP-2 under thecontrol of a strong promoter system), and transgenic animals thatexpress a MASP-2 transgene.

[0045] The invention encompasses methods for treating disordersassociated with aberrant expression or activity of MASP-2. Thus, theinvention includes methods for treating disorders associated withexcessive expression or activity of MASP-2. Such methods entailadministering a compound which decreases the expression or activity ofMASP-2. The invention also includes methods for treating disordersassociated with insufficient expression of MASP-2. These methods entailadministering a compound which increases the expression or activity ofMASP-2.

[0046] By “competitively inhibiting” serine protease activity is meantthat, for example, the action of an altered MBL or fragment thereof thatcan bind to a MASP-2 peptide, reversibly or irreversibly withoutactivating serine protease activity. Conversely, a fragment of MASP-2,e.g., a polypeptide encompassing the N-terminal part of MASP-2, maycompetitively inhibit the binding of the intact MASP-2 and thuseffectively inhibit the activation of MASP-2.

[0047] The invention also features methods for detecting a MASP-2polypeptide. Such methods include: obtaining a biological sample;contacting the sample with an antibody that specifically binds MASP-2under conditions which permit specific binding; and detecting anyantibody-MASP-2 complexes formed.

[0048] In addition, the present invention encompasses methods andcompositions for the diagnostic evaluation, typing, and prognosis ofdisorders associated with inappropriate expression or activity ofMASP-2. For example, the nucleic acid molecules of the invention can beused as diagnostic hybridization probes to detect, for example,inappropriate expression of MASP-2 or mutations in the MASP-2 gene. Suchmethods may be used to classify cells by the level of MASP-2 expression.

[0049] Alternatively, the nucleic acid molecules can be used as primersfor diagnostic PCR analysis for the identification of gene mutations,allelic variations and regulatory defects in the MASP-2 gene. Thepresent invention further provides for diagnostic kits for the practiceof such methods.

[0050] The invention features methods of identifying compounds thatmodulate the expression or activity of MASP-2 by assessing theexpression or activity of MASP-2 in the presence and absence of aselected compound. A difference in the level of expression or activityof MASP-2 in the presence and absence of the selected compound indicatesthat the selected compound is capable of modulating expression oractivity or MASP-2. Expression can be assessed either at the level ofgene expression (e.g., by measuring mRNA) or protein expression bytechniques that are well known to skilled artisans. The activity ofMASP-2 can be assessed functionally, i.e., by assaying the ability ofthe compound to activate complement.

[0051] The preferred methods and materials are described below inexamples which are meant to illustrate, not limit, the invention.Skilled artisans will recognize methods and materials that are similaror equivalent to those described herein, and that can be used in thepractice or testing of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described herein. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andare not intended to be limiting.

[0053] Other features and advantages of the invention will be apparentfrom the detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054]FIGS. 1a-1 b depict a Western blot of human plasma proteinspurified by sugar affinity chromatography.

[0055]FIG. 2 shows the sequence alignments of the amino acid sequencesof MASP-2 (clone phl-4) , MASP-1^(17,22), C1r^(23,24) and C1s^(25,26).

[0056]FIGS. 3a-3 b are representations of the results demonstratingmolecular complexes formed between MBL, MASP-1 and MASP-2.

[0057]FIGS. 4a-4 b are representations of Western blots demonstratingthe activation of C4 by C1s and MASP-2.

[0058]FIG. 5 illustrates the three pathways of complement activation.

[0059]FIG. 6 shows the cDNA sequence and deduced amino acid sequence ofMASP-2.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] MASP-2 Nucleic Acid Molecules

[0061] The MASP-2 nucleic acid molecules of the invention can be cDNA,genomic DNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded (i.e., either a sense or an antisense strand). Fragmentsof these molecules are also considered within the scope of theinvention, and can be produced, for example, by the polymerase chainreaction (PCR) or generated by treatment with one or more restrictionendonucleases. A ribonucleic acid (RNA) molecule can be produced by invitro transcription. Preferably, the nucleic acid molecules encodepolypeptides that, regardless of length, are soluble under normalphysiological conditions.

[0062] The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (for example, the polypeptide of SEQ ID NO:2). Inaddition, these nucleic acid molecules are not limited to sequences thatonly encode polypeptides, and thus, can include some or all of thenon-coding sequences that lie upstream or downstream from a codingsequence.

[0063] The nucleic acid molecules of the invention can be synthesized(for example, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, mouse, rat, guinea pig, cow, sheep, horse, pig,rabbit, monkey, dog, or cat. Combinations or modifications of thenucleotides within these types of nucleic acids are also encompassed.

[0064] In addition, the isolated nucleic acid molecules of A theinvention encompass fragments that are not found as such in the naturalstate. Thus, the invention encompasses recombinant molecules, such asthose in which a nucleic acid molecule (for example, an isolated nucleicacid molecule encoding MASP-2) is incorporated into a vector (forexample, a plasmid or viral vector) or into the genome of a heterologouscell (or the genome of a homologous cell, at a position other than thenatural chromosomal location). Recombinant nucleic acid molecules anduses therefore are discussed further below.

[0065] In the event the nucleic acid molecules of the invention encodeor act as antisense molecules, they can be used for example, to regulatetranslation of MASP-2. Techniques associated with detection orregulation of nucleic acid expression are well known to skilled artisansand can be used to diagnose and/or treat disorders associated withMASP-2 activity. These nucleic acid molecules are discussed furtherbelow in the context of their clinical utility.

[0066] The invention also encompasses nucleic acid molecules thathybridize under stringent conditions to a nucleic acid molecule encodinga MASP-2 polypeptide. The cDNA sequence described herein (SEQ ID NO:3)can be used to identify these nucleic acids, which include, for example,nucleic acids that encode homologous polypeptides in other species, andsplice variants of the MASP-2 gene in humans or other mammals.Accordingly, the invention features methods of detecting and isolatingthese nucleic acid molecules. Using these methods, a sample (forexample, a nucleic acid library, such as a cDNA or genomic library) iscontacted (or “screened”) with a MASP-2-specific probe (for example, afragment of SEQ ID NO:3 that is at least 12 nucleotides long). The probewill selectively hybridize to nucleic acids encoding relatedpolypeptides (or to complementary sequences thereof). Because thepolypeptide encoded by MASP-2 is related to other serine proteases, theterm “selectively hybridize” is used to refer to an event in which aprobe binds to nucleic acids encoding MASP-2 (or to complementarysequences thereof) to a detectably greater extent than to nucleic acidsencoding other serine proteases (or to complementary sequences thereof).The probe, which can contain at least 12 (for example, 15, 25, 50, 100,or 200 nucleotides) can be produced using any of several standardmethods (see, for example, Ausubel et al.,“Current Protocols inMolecular Biology, Vol. I,” Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., NY, 1989). For example, the probe can be generatedusing PCR amplification methods in which oligonucleotide primers areused to amplify a MASP-2-specific nucleic acid sequence (for example, anucleic acid encoding the N-terminus of mature MASP-2) that can be usedas a probe to screen a nucleic acid library, as described in Example 4below, and thereby detect nucleic acid molecules (within the library)that hybridize to the probe.

[0067] One single-stranded nucleic acid is said to hybridize to anotherif a duplex forms between them. This occurs when one nucleic acidcontains a sequence that is the reverse and complement of the other(this same arrangement gives rise to the natural interaction between thesense and antisense strands of DNA in the genome and underlies theconfiguration of the “double helix”). Complete complementarity betweenthe hybridizing regions is not required in order for a duplex to form;it is only necessary that the number of paired bases is sufficient tomaintain the duplex under the hybridization conditions used.

[0068] Typically, hybridization conditions are of low to moderatestringency. These conditions favor specific interactions betweencompletely complementary sequences, but allow some non-specificinteraction between less than perfectly matched sequences to occur aswell. After hybridization, the nucleic acids can be “washed” undermoderate or high conditions of stringency to dissociate duplexes thatare bound together by some non-specific interaction (the nucleic acidsthat form these duplexes are thus not completely complementary).

[0069] As is known in the art, the optimal conditions for washing aredetermined empirically, often by gradually increasing the stringency.The parameters that can be changed to affect stringency include,primarily, temperature and salt concentration. In general, the lower thesalt concentration and the higher the temperature, the higher thestringency. Washing can be initiated at a low temperature (for example,room temperature) using a solution containing a salt concentration thatis equivalent to or lower than that of the hybridization solution.Subsequent washing can be carried out using progressively warmersolutions having the same salt concentration. As alternatives, the saltconcentration can be lowered and the temperature maintained in thewashing step, or the salt concentration can be lowered and thetemperature increased. Additional parameters can also be altered. Forexample, use of a destabilizing agent, such as formamide, alters thestringency conditions.

[0070] In reactions where nucleic acids are hybridized, the conditionsused to achieve a given level of stringency will vary. There is not oneset of conditions, for example, that will allow duplexes to form betweenall nucleic acids that are 85% identical to one another; hybridizationalso depends on unique features of each nucleic acid. The length of thesequence, the composition of the sequence (for example, the content ofpurine-like nucleotides versus the content of pyrimidine-likenucleotides) and the type of nucleic acid (for example, DNA or RNA)affect hybridization. An additional consideration is whether one of thenucleic acids is immobilized (for example, on a filter).

[0071] An example of a progression from lower to higher stringencyconditions is the following, where the salt content is given as therelative abundance of SSC (a salt solution containing sodium chlorideand sodium citrate; 2×SSC is 10-fold more concentrated than 0.2×SSC).Nucleic acids are hybridized at 42° C. in 2×SSC/0.1% SDS (sodiumdodecylsulfate; a detergent) and then washed in 0.2×SSC/0.1% SDS at roomtemperature (for conditions of low stringency); 0.2×SSC/0.1% SDS at 42°C. (for conditions of moderate stringency); and 0.1×SSC at 68° C. (forconditions of high stringency). Washing can be carried out using onlyone of the conditions given, or each of the conditions can be used (forexample, washing for 10-15 minutes each in the order listed above). Anyor all of the washes can be repeated. As mentioned above, optimalconditions will vary and can be determined empirically.

[0072] A second set of conditions that are considered “stringentconditions” are those in which hybridization is carried out at 50° C. inChurch buffer (7% SDS, 0.5% NaHPO₄, 1 M EDTA, 1% bovine serum albumin)and washing is carried out at 50° C. in 2×SSC.

[0073] Once detected, the nucleic acid molecules can be isolated by anyof a number of standard techniques (see, for example, Sambrook et al.,“Molecular Cloning, A Laboratory Manual,” 2nd Ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989).

[0074] The invention also encompasses: (a) expression vectors thatcontain any of the foregoing MASP-2-related coding sequences and/ortheir complements (that is, “antisense” sequence); (b) expressionvectors that contain any of the foregoing MASP-2-related codingsequences operatively associated with a regulatory element (examples ofwhich are given below) that directs the expression of the codingsequences; (c) expression vectors containing, in addition to sequencesencoding a MASP-2 polypeptide, nucleic acid sequences that are unrelatedto nucleic acid sequences encoding MASP-2, such as molecules encoding areporter or marker; and (d) genetically engineered host cells thatcontain any of the foregoing expression vectors and thereby express thenucleic acid molecules of the invention in the host cell.

[0075] Recombinant nucleic acid molecule can contain a sequence encodinga soluble MASP-2, mature MASP-2, MASP-2 having a signal sequence, orfunctional domains of MASP-2 such as the serine protease domain, EGFdomain, or the MBL-binding domain. The full length MASP-2 polypeptide, adomain of MASP-2, or a fragment thereof may be fused to additionalpolypeptides, as described below. Similarly, the nucleic acid moleculesof the invention can encode the mature form of MASP-2 or a form thatencodes a polypeptide which facilitates secretion. In the latterinstance, the polypeptide is typically referred to as a proprotein,which can be converted into an active form by removal of the signalsequence, for example, within the host cell. Proproteins can beconverted into the active form of the protein by removal of theinactivating sequence.

[0076] The regulatory elements referred to above include, but are notlimited to, inducible and non-inducible promoters, enhancers, operatorsand other elements, which are known to those skilled in the art, andwhich drive or otherwise regulate gene expression. Such regulatoryelements include but are not limited to the cytomegalovirus hCMVimmediate early gene, the early or late promoters of SV40 adenovirus,the lac system, the trp system, the TAC system, the TRC system, themajor operator and promoter regions of phage A, the control regions offd coat protein, the promoter for 3-phosphoglycerate kinase, thepromoters of acid phosphatase, and the promoters of the yeast α-matingfactors.

[0077] Similarly, the nucleic acid can form part of a hybrid geneencoding additional polypeptide sequences, for example, sequences thatfunction as a marker or reporter. Examples of marker or reporter genesinclude β-lactamase, chloramphenicol acetyltransferase (CAT), adenosinedeaminase (ADA), aminoglycoside phosphotransferase (neo^(r), G418^(r)),dihydrofolate reductase (DHFR), hygromycin-B-phospohotransferase (HPH),thymidine kinase (TK), lacZ (encoding β-galactosidase) green fluorescentprotein (GFP), and xanthine guanine phosphoribosyltransferase (XGPRT).As with many of the standard procedures associated with the practice ofthe invention, skilled artisans will be aware of additional usefulreagents, for example, of additional sequences that can serve thefunction of a marker or reporter. Generally, the hybrid polypeptide willinclude a first portion and a second portion; the first portion being aMASP-2 polypeptide and the second portion being, for example, thereporter described above or an immunoglobulin constant region.

[0078] The expression systems that may be used for purposes of theinvention include, but are not limited to, microorganisms such asbacteria (for example, E. coli and B. subtilis) transformed withrecombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expressionvectors containing the nucleic acid molecules of the invention; yeast(for example, Saccharomyces and Pichia) transformed with recombinantyeast expression vectors containing the nucleic acid molecules of theinvention (preferably containing the nucleic acid sequence of MASP-2(SEQ ID NO:3)) ; insect cell systems infected with recombinant virusexpression vectors (for example, baculovirus) containing the nucleicacid molecules of the invention; plant cell systems infected withrecombinant virus expression vectors (for example, cauliflower mosaicvirus (CaMV) and tobacco mosaic virus (TMV)) or transformed withrecombinant plasmid expression vectors (for example, Ti plasmid)containing MASP-2 nucleotide sequences; or mammalian cell systems (forexample, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (for example, the metallothioneinpromoter) or from mammalian viruses (for example, the adenovirus latepromoter and the vaccinia virus 7.5K promoter).

[0079] In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions containing MASP-2 polypeptides or for raising antibodies tothose polypeptides, vectors that are capable of directing the expressionof high levels of fusion protein products that are readily purified maybe desirable. Such vectors include, but are not limited to, the E. coliexpression vector pUR278 (Ruther et al., EMBO J. 2:1791, 1983), in whichthe coding sequence of the insert may be ligated individually into thevector in frame with the lacZ coding region so that a fusion protein isproduced; pIN vectors (Inouye and Inouye, Nucleic Acids Res.13:3101-3109, 1985; Van Heeke and Schuster, J. Biol. Chem.264:5503-5509, 1989); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

[0080] In an insect system, Autographa californica nuclear polyhidrosisvirus (AcNPV) can be used as a vector to express foreign genes. Thevirus grows in Spodoptera frugiperda cells. The coding sequence of theinsert may be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter). Successfulinsertion of the coding sequence will result in inactivation of thepolyhedrin gene and production of non-occluded recombinant virus (i.e.,virus lacking the proteinaceous coat coded for by the polyhedrin gene).These recombinant viruses are then used to infect Spodoptera frugiperdacells in which the inserted gene is expressed. (for example, see Smithet al., J. Virol. 46:584, 1983; Smith, U.S. Pat. No. 4,215,051).

[0081] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, the nucleic acid molecule of the invention may beligated to an adenovirus transcription/translation control complex, forexample, the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (for example, region E1 or E3) will result in a recombinant virusthat is viable and capable of expressing a MASP-2 gene product ininfected hosts (for example, see Logan and Shenk, Proc. Natl. Acad. Sci.USA 81:3655-3659, 1984). Specific initiation signals may also berequired for efficient translation of inserted nucleic acid molecules.These signals include the ATG initiation codon and adjacent sequences.In cases where an entire gene or cDNA, including its own initiationcodon and adjacent sequences, is inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only a portion of the coding sequence isinserted, exogenous translational control signals, including, perhaps,the ATG initiation codon, must be provided. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:516-544, 1987).

[0082] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (forexample, glycosylation) and processing (for example, cleavage) ofprotein products may be important for the function of the protein.Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins and geneproducts. Appropriate cell lines or host systems can be chosen to ensurethe correct modification and processing of the foreign proteinexpressed. To this end, eukaryotic host cells which possess the cellularmachinery for proper processing of the primary transcript,glycosylation, and phosphorylation of the gene product may be used. Themammalian cell types listed above are among those that could serve assuitable host cells.

[0083] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress the MASP-2 sequences described above may be engineered. Ratherthan using expression vectors which contain viral origins ofreplication, host cells can be transformed with DNA controlled byappropriate expression control elements (for example, promoter, enhancersequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. Following the introduction of the foreign DNA,engineered cells may be allowed to grow for 1-2 days in an enrichedmedia, and then switched to a selective media. The selectable marker inthe recombinant plasmid confers resistance to the selection and allowscells to stably integrate the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines. Thismethod can advantageously be used to engineer cell lines which expressMASP-2. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the gene product and for production of MASP-2 for theraputicuses. These methods may also be used to modify cells that are introducedinto a host organism either for experimental or theraputic purposes. Theintroduced cells may be transient or permanent within the host organism.

[0084] A number of selection systems can be used. For example, theherpes simplex virus thymidine kinase (Wigler, et al., Cell 11:223,1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska andSzybalski, Proc. Naati. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,anti-metabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Proc. Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc.Natl. Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA78:2072, 1981); neo, which confers resistance to the aminoglycosideG-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147,1984).

[0085] Alternatively, any fusion protein may be readily purified byutilizing an antibody specific for the fusion protein being expressed.For example, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Proc. Natl. Acad. Sci. USA 88: 8972-8976, 1991). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the gene's open reading frame is translationally fused to anamino-terminal tag consisting of six histidine residues. Extracts fromcells infected with recombinant vaccinia virus are loaded ontoNi²⁺.nitriloacetic acid-agarose columns and histidine-tagged proteinsare selectively eluted with imidazole-containing buffers.

[0086] MASP-2 Polypeptides

[0087] The MASP-2 polypeptides described herein are those encoded by anyof the nucleic acid molecules described above and include MASP-2fragments, mutants, truncated forms, and fusion proteins. Thesepolypeptides can be prepared for a variety of uses, including but notlimited to the generation of antibodies, as reagents in diagnosticassays, for the identification of other cellular gene products orcompounds that can modulate the MBLectin response, and as pharmaceuticalreagents useful for the treatment of inflammation and certain disorders(described below) that are associated with activity of of the MBLectinpathway. Preferred polypeptides are substantially pure MASP-2polypeptides, including those that correspond to the polypeptide with anintact signal sequence (extending from amino acids 1-15 of SEQ ID NO:2),the mature form of the polypeptide (extending from amino acids 16-686 ofSEQ ID NO:2) of the human MASP-2 polypeptide as well as polypeptidesrepresenting a part of the MASP-2 polypeptide. Especially preferred arepolypeptides that are soluble under normal physiological conditions.

[0088] The invention also encompasses polypeptides that are functionallyequivalent to MASP-2. These polypeptides are equivalent to MASP-2 inthat they are capable of carrying out one or more of the functions ofMASP-2 in a biological system. Preferred MASP-2 polypeptides have 20%,40%, 50%, 75%, 80%, or even 90% of the activity of the full-length,mature human form of MASP-2 described herein. Such comparisons aregenerally based on an assay of biological activity in which equalconcentrations of the polypeptides are used and compared. The comparisoncan also be based on the amount of the polypeptide required to reach 50%of the maximal activity obtainable.

[0089] Functionally equivalent proteins can be those, for example, thatcontain additional or substituted amino acid residues. Substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. Amino acids that are typically considered to providea conservative substitution for one another are specified in the summaryof the invention. D-amino acids may be introduced in order to modify thehalf-life of the polypeptide.

[0090] Polypeptides that are functionally equivalent to MASP-2 (SEQ IDNO:2) can be made using random mutagenesis techniques well known tothose skilled in the art (and the resulting mutant MASP-2 proteins canbe tested for activity). It is more likely, however, that suchpolypeptides will be generated by site-directed mutagenesis (again usingtechniques well known to those skilled in the art). These polypeptidesmay have an increased function, i.e., a greater ability to activate theMBLectin pathway. Such polypeptides can be used to enhance the activityof MBLectin pathway immune function.

[0091] To design functionally equivalent polypeptides, it is useful todistinguish between conserved positions and variable positions. This canbe done by aligning the sequence of MASP-2 cDNAs that were obtained fromvarious organisms. Skilled artisans will recognize that conserved aminoacid residues are more likely to be necessary for preservation offunction. Thus, it is preferable that conserved residues are notaltered.

[0092] Mutations within the MASP-2 coding sequence can be made togenerate MASP-2 peptides that are better suited for expression in aselected host cell. Introduction of a glycosylation sequence can also beused to generate a MASP-2 polypeptide with altered biologicalcharacteristics.

[0093] The invention also features methods for assay of polymorphismswithin the polypeptide sequence comprising MASP-2 or its precursor. Thismay be accomplished by a number of techniques. For example, the purifiedpolypeptide is subjected to tryptic digestion and the resultingfragments analyzed by either one-or two dimensional electrophoresis. Theresults from analysis of a sample polypeptide are compared to theresults using a known sequence. Also the analysis may encompassseparation of a biological sample (e.g., serum or other body fluids) byeither one- or two-dimensional electrophoresis followed by transfer ofthe separated proteins onto a membrane (western blot). The membrane isthen reacted with antibodies against MASP-2, followed by a secondarylabelled antibody. The staining pattern is compared with that obtainedusing a sample with a known sequence or modification.

[0094] The polypeptides of the invention can be expressed fused toanother polypeptide, for example, a marker polypeptide or fusionpartner. For example, the polypeptide can be fused to a hexa-histidinetag to facilitate purification of bacterially expressed protein or ahemagglutinin tag to facilitate purification of protein expressed ineukaryotic cells. The MASP-2 polypeptide of the invention, or a portionthereof, can also be altered so that it has a longer circulatinghalf-life by fusion to an immunoglobulin Fc domain (Capon et al., Nature337:525-531, 1989). Similarly, a dimeric form of the MASP-2 polypeptidecan be produced, which has increased stability in vivo.

[0095] The polypeptides of the invention can be chemically synthesized(for example, see Creighton, “Proteins: Structures and MolecularPrinciples,” W. H. Freeman & Co., NY, 1983), or, perhaps moreadvantageously, produced by recombinant DNA technology as describedherein. For additional guidance, skilled artisans may consult Ausubel etal. (supra), Sambrook et al. (“Molecular Cloning, A Laboratory Manual,”Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989), and,particularly for examples of chemical synthesis Gait, M. J. Ed.(“Oligonucleotide Synthesis,” IRL Press, Oxford, 1984).

[0096] The invention also features polypeptides that interact withMASP-2 (and the genes that encode them) and thereby alter the functionof MASP-2 interacting polypeptides can be identified using methods knownto those skilled in the art. One suitable method is the “two-hybridsystem,” which detects protein interactions in vivo (Chien et al., Proc.Natl. Acad. Sci. USA, 88:9578, 1991). A kit for practicing this methodis available from Clontech (Palo Alto, Calif.).

[0097] Anti-MASP-2 Antibodies

[0098] Human MASP-2 polypeptides (or immunogenic fragments or analogs)can be used to raise antibodies useful in the invention; suchpolypeptides can be produced by recombinant techniques or synthesized(see, for example, “Solid Phase Peptide Synthesis,” supra; Ausubel etal., supra) . In general, the peptides can be coupled to a carrierprotein, such as KLH, as described in Ausubel et al., supra, mixed withan adjuvant, and injected into a host mammal. Also the carrier could bePPD. Antibodies can be purified by peptide antigen affinitychromatography.

[0099] In particular, various host animals can be immunized by injectionwith a MASP-2 protein or polypeptide. Host animals include rabbits,mice, guinea pigs, rats, and chickens. Various adjuvants that can beused to increase the immunological response depend on the host speciesand include Freund's adjuvant (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Potentially useful humanadjuvants include BCG (bacille Calmette-Guerin) and Corynebacteriumparvum. Polyclonal antibodies are heterogeneous populations of antibodymolecules that are contained in the sera of the immunized animals.

[0100] Antibodies within the invention therefore include polyclonalantibodies and, in addition, monoclonal antibodies, humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, and molecules produced using a Fab expression library, andantibodies or fragments produced by phage display techniques.

[0101] Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be prepared using the MASP-2proteins described above and standard hybridoma technology (see, forexample, Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J.Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976;Hammerling et al., “Monoclonal Antibodies and T Cell Hybridomas,”Elsevier, N.Y., 1981; Ausubel et al., supra).

[0102] In particular, monoclional antibodies can be obtained by anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture such as described in Kohler et al.,Nature 256:495, 1975, and U.S. Pat. No. 4,376,110; the human B-cellhybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole etal., Proc. Natl. Acad. Sci. USA 80:2026, 1983), and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy,” AlanR. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclassthereof. (In the case of chckens, the immunoglobulin class can also beIgY.) The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. The ability to produce high titers ofmabs in vivo makes this the presently preferred method of production,but in some cases, in vitro production will be preferred to avoidintroducing cancer cells into live animals, for example, in cases wherethe presence of normal immunoglobulins coming from the acitis fluids areunwanted, or in cases involving ethical considerations.

[0103] Once produced, polyclonal, monoclonal, or phage-derivedantibodies are tested for specific MASP-2 recognition by Western blot orimmunoprecipitation analysis by standard methods, e.g., as described inAusubel et al., supra. Antibodies that specifically recognize and bindto MASP-2 are useful in the invention. For example, such antibodies canbe used in an immunoassay to monitor the level of MASP-2 produced by ananimal (for example, to determine the amount or subcellular location ofMASP-2).

[0104] Preferably, antibodies of the invention are produced usingfragments of the MASP-2 protein which lie outside highly conservedregions and appear likely to be antigenic, by criteria such as highfrequency of charged residues. In one specific example, such fragmentsare generated by standard techniques of PCR, and are then cloned intothe pGEX expression vector (Ausubel et al., supra). Fusion proteins areexpressed in E. coli and purified using a glutathione agarose affinitymatrix as described in Ausubel, et al., supra.

[0105] In some cases it may be desirable to minimize the potentialproblems of low affinity or specificity of antisera. In suchcircumstances, two or three fusions can be generated for each protein,and each fusion can be injected into at least two rabbits. Antisera canbe raised by injections in a series, preferably including at least threebooster injections.

[0106] Antisera is also checked for its ability to immunoprecipitaterecombinant MASP-2 proteins or control proteins, such as glucocorticoidreceptor, CAT, or luciferase.

[0107] The antibodies can be used, for example, in the detection of theMASP-2 in a biological sample as part of a diagnostic assay. Antibodiesalso can be used in a screening assay to measure the effect of acandidate compound on expression or localization of MASP-2.Additionally, such antibodies can be used in conjunction with the genetherapy techniques described to, for example, evaluate the normal and/orengineered MASP-2-expressing cells prior to their introduction into thepatient. Such antibodies additionally can be used in a method forinhibiting abnormal MASP-2 activity.

[0108] In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851, 1984;Neuberger et al., Nature, 312:604, 1984; Takeda et al., Nature, 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region.

[0109] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692)can be adapted to produce single chain antibodies against a MASP-2protein or polypeptide. Single chain antibodies are formed by linkingthe heavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

[0110] Antibody fragments that recognize and bind to specific epitopescan be generated by known techniques. For example, such fragmentsinclude but are not limited to F(ab′)₂ fragments that can be produced bypepsin digestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science, 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

[0111] Antibodies to MASP-2 can, in turn, be used to generateanti-idiotype antibodies that resemble a portion of MASP-2 usingtechniques well known to those skilled in the art (see, e.g., Greenspanet al., FASEB J. 7:437, 1993; Nissinoff, J. Immunol. 147:2429, 1991).For example, antibodies that bind to MASP-2 and competitively inhibitthe binding of a ligand of MASP-2 can be used to generate anti-idiotypesthat resemble a ligand binding domain of MASP-2 and, therefore, bind andneutralize a ligand of MASP-2 such as MBL. Such neutralizinganti-idiotypic antibodies or Fab fragments of such anti-idiotypicantibodies can be used in therapeutic regimens.

[0112] Antibodies can be humanized by methods known in the art. Forexample, monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics 7:13-21, 1994; see also U.S. Pat. Nos. 5,545,806 and 5,569,825,both of which are hereby incorporated by reference).

[0113] The methods described herein in which anti-MASP-2 antibodies areemployed may be performed, for example, by utilizing pre-packageddiagnostic kits comprising at least one specific MASP-2 nucleotidesequence or antibody reagent described herein, which may be convenientlyused, for example, in clinical settings, to diagnose patients exhibitingsymptoms of the disorders described below.

[0114] Quantitative Assays of MASP-2

[0115] As an example only, quantitative assays may be devised for theestimation of MASP-2 concentrations in body fluids or organ (biopsy)extracts. Such assays may be fluid phase or solid phase. Examples arecompetitive and non-competitive ELISAs. As an example of the latter,microtiter wells are coated with anti-MASP-2 antibody, incubated withsamples, and the presence of MASP-2 visualized with enzyme-labelledantibody followed by substrate that deposits a colored compound.Alternatively, a label such as europium may be used and the detectionmade by use of time resolved fluorometry.

[0116] Assays of the functional activity of MASP-2, either alone or aspart of the MBL/MASP complex may be performed by several methods. As anexample of a test for MBL/MASP-2 complex, the test sample is appliedonto mannan-coated micro wells and C4 is added to estimate theC4-cleaving activity, or C3 is added to estimate the C3 cleavingactivity of the generated C3 convertase. Assay of MASP-2 not occurringas part of the MBL/MASP complex is carried out similarly, but MBL isadded either to the micro well or to the sample before adding this tothe mannan-coated well. Before the addition of MBL the sample may bedepleted of MBL and MBL/MASP-1 and MBL/MASP-2 complexes by treatmentwith solid phase mannan, e.g. attached to beads, or by solid phaseanti-MBL antibodies, or by treatment with a suitable concentration of aprecipitating agent, e.g., PEG, which precipitates the complex butleaves MASP-2 in the supernatant. The assay is carried out at conditionswhich minimize or eliminate interference from the classical complementactivation pathway and the alternative complement activation pathway.

[0117] Assays estimating the activity of MASP-2 or MASP-2 may be usedfor diagnostic and treatment purposes in samples from individuals,notably those suffering from infectious or inflammatory diseases.

[0118] MASP-2 for Therapy

[0119] Therapeutic use of components specified in the claims may beapplied in situations where a constitutional or temporary deficiency inMASP-2 renders the individual susceptible to one or more infections, orsituations where the individual cannot neutralize an establishedinfection. MASP-2 or MBL/MASP complexes can be administered, preferablyby intravenous infusions, in order to improve the individual's immunedefense.

[0120] We believe MASP-2 is required for the powerful antimicrobialactivity of the MBL/MASP complex, and deficiency in MASP-2, eithergenetically determined or acquired, will therefore compromise anindividual's resistance to infections and ability to combat establishedinfections. Reconstitution with natural or recombinant MASP-2 is auseful treatment modality in such situations. Recombinant MASP-2 may bein the form of the whole molecule, parts of the molecule, or the wholeor part thereof attached by any means to another structure in order tomodulate the activity. The recombinant products may be identical instructure to the natural molecule or slightly modified to yield enhancedactivity or decreased activity when such is desired.

[0121] Reconstitution therapy with MBL, either natural or recombinant,requires that the recipient has sufficient MASP-2 for the expression ofMBL/MASP activity. Thus, MASP-2 must be included in the therapeuticpreparation when the patient has insufficient MASP-2 activity.

[0122] Assays for MASP-2

[0123] Therapy with MASP-2 (or MASP-2 inhibitors) must usually bepreceded by the estimation of MASP-2 in serum or plasma from thepatient. Examples of such assays are described below.

[0124] Assays for MASP-2 Antigen.

[0125] MASP-2 protein is conveniently estimated as antigen using one ofthe standard immunological procedures.

[0126] As an example only, a quantitative TRIFMA (time resolvedimmunofluorometric assay) for MASP-2 was constructed by 1) coatingmicrotiter wells with 1 μg anti-C′ MASP-2 antibody; 2) blocking withTween-20; 3) applying test samples, e.g. diluted plasma or serumsamples: 4) applying Eu-labelled anti-N′ MASP-2 antibody; 5) applyingenhancement solution (Wallac Ltd): 6) reading the Eu on a time resolvedfluorometer. (Estimation by ELISA may be carried out similarly, e.g. byusing biotin-labelled anti-N′ MASP-2 in step 4; alkalinephosphatase-labelled avidin in step 5; 6) apply substrate; and 7) readthe colour intensity.) Between each step, the plate was incubated atroom temperature and washed, except between step 6 and 7. A calibrationcurve may be constructed using dilutions of pooled normal plasma,arbitrarily said to contain 1 unit of MASP-2 per ml. The antibodies usedin this first version of a MASP-2 assay were raised against syntheticpeptides and reacted poorly with native MASP-2. The samples are thuspretreated with SDS on a boiling water bath for 5 min. and the SDSneutralized with non-ionic detergent (Triton X-100) before the assay. Afurther development of the assay employs antibodies reacting with nativeMASP-2, thus rendering the SDS treatment superfluous.

[0127] Assays may be similarly constructed using antibodies, polyclonalor monoclonal or recombinant antibodies, which reacts with MASP-2,natural or recombinant, or parts thereof.

[0128] Through the use of antibodies reacting selectively with intactMASP-2 or with activation products, or through combination of antibodiesagainst various parts of the molecule, assays may be constructed for theestimation of the activation in vivo of the MBLectin pathway. Theseassays will be useful for the determination of inflammation caused bythe activation of this pathway.

[0129] Assays for MASP-2 Activity of the MPL/MASP Complex.

[0130] MASP-2 may be estimated by its capacity to activate thecomplement system. When C4 is cleaved by MASP-2 an active thiol ester isexposed and C4 becomes covalently attached to nearby nucleophilicgroups. A substantial part of the C4b will thus become attached to thecoated plastic well and may be detected by anti-C4 antibody. Aquantitative TRIFMA for MASP-2 activity was constructed by 1) coatingmicrotiter wells with 1 μg mannan in 100 μl buffer; 2) blocking withTween-20; 3) applying test samples, e.g. diluted plasma or serumsamples: 4) applying purified complement factor C4 at 5 μg/ml; 5)incubate for one hour at 37° C.; 6) applying Eu-labelled anti-C4antibody; 7) applying enhancement solution; and 8) reading the Eu bytime resolved fluorometry. (Estimation by ELISA may be carried outsimilarly, e.g. by applying biotin-labelled anti-C4 in step 6; 7) applyalkaline phosphatase-labelled avidin; 8) apply substrate; and 9) readthe colour intensity). Between each step the plate was incubated at roomtemperature and washed, except between step 7 and 8. A calibration curvecan be constructed using dilutions of one selected normal plasma,arbitrarily said to contain 1 unit of MBL/MASP-2 activity per ml. Theassay is carried out at conditions which preclude activation of C4 bythe classical or alternative complement activation pathways. Theactivation of C4 was completely inhibited by the serine proteaseinhibitor benzamidine. Activation of the classical pathway iseffectively eliminated by carrying out step 3) in the presence ofsufficiently high ionic strength (0.7 to 2.0 NaCl; preferably about 1.0M NaCl) which does not interfere with the MBL/MASP complex butcompletely destroys the C1qrs complex; activation of the alternativepathway is effectively precluded by assaying at a dilution of 20-fold orgreater.

[0131] Assays for the Estimation of Free MASP-2 Activity.

[0132] The estimation of MASP-2 activity in samples from MBL-deficientindividuals is carried out on wells coated with MASP-free MBL. Theestimation of free MASP in samples from individuals with MBL is carriedout by first removing MBL/MASP-1 and MBL/MASP-2 complexes by incubatingwith Sepharose-coupled mannan (300 μl of 10 fold diluted plasma or serumis incubated with 10 μl beads), and then analyzing the supernatant.

[0133] The assay carried out in the TRIFMA formate proceeds asfollows: 1) coating microtiter wells with 1 μg mannan in 100 μl buffer;2) blocking with Tween-20; 3) incubate sample at a 1000 fold dilution inbuffer with 100 ng of MASP-free MBL/ml, and applying 100 μl of themixture per well; 4) incubate over night at 4° C.; 4) wash and applyingpurified complement factor C4 at 5 μg/ml; 5) incubate for one hour at37° C.; 6) applying Eu-labelled anti-C4 antibody; 7) applyingenhancement solution; and 8) reading the Eu by time resolvedfluorometry. (Estimation by ELISA may be carried out similarly, e.g. byapplying biotin-labelled anti-C4 in step 6; 7) apply alkalinephosphatase-labelled avidin; 8) apply substrate; and 9) read the colourintensity.) Between each step the plate was washed, except between step7 and 8. A calibration curve may be constructed using dilutions of oneselected MBL-deficient plasma, arbitrarily said to contain 1 unit ofMASP-2 activity per ml. The assay is carried out at conditions whichpreclude activation of C4 by the classical or alternative complementactivation pathways (see above).

[0134] Inhibition of MASP-2 Activity.

[0135] Inhibitors of the biological activity of MASP-2 may be employedto control the complement activating activity and inflammatory activityof MASP-2. Such inhibitors may be substrate analogues representingtarget structures of C2 or C4. Inhibitors may be of peptide nature,modified peptides, or any organic molecule which inhibits the activityof MASP-2 competitively or non-competitively. The inhibitor may bemodified to stay in circulation for short or longer time, andconstructed to be given by injection or perorally. Inhibitors may befragments of MASP-2, produced from natural or recombinant MASP-2, bychemical or enzymatic procedures. Inhibitors may be naturally occurringshorter forms of MASP-2. Inhibitors may be in soluble form or coupled toa solid phase. A solid phase could be a compatible surface such as usedin extracorporal blood or plasma flow devices.

[0136] Microbial carbohydrates or endogenous oligosaccharides mayprovoke undesirable activation of the MBL/MASP complex resulting indamaging inflammatory responses. This pathophysiological activity may bereduced though the administration of inhibitors of MASP-2 activity suchas Pefabloc. Also other enzyme inhibitors (PMSF, benzamidine, etc.) haveproved effective when assayed in the TRIFMA for MASP-2 activity.Obviously, when designing inhibitors for in vivo use toxicity is a majorconsideration, and highly specific inhibitors can be assumed to be lesstoxic than more broadly reactive inhibitors. Specific inhibitors may begenerated through using peptides, peptide analogues or peptidederivatives representing the target structures on complement factor C4or C2 molecules. Another type of inhibitors may be based on antibodies(or fragments of antibodies) against the active site of MASP-2 or otherstructures on MASP-2 thus inhibiting the activity of MASP-2. Inhibitorsmay also be directed towards inhibition of the activation of MASP-2,thought to be effected by MASP-1, i.e. the target structure for MASP-1on MASP-2 would be a suitable inhibitor of this type. Another type ofinhibitor would prevent the binding of MASP-2 to MBL and thereby theactivation of MASP-2. The N-terminal 20 kDa fragment of MASP-2 may be asuitable inhibitor of this type. More specifically one can localize theprecise part of the polypeptide chain which mediates the binding ofMASP-2 to MBL and use the synthetic peptide or analogous structures asinhibitor. Inhibitors may be substituted with D amino acids for L-aminoacids.

[0137] Also, inhibitors could be RNA or single stranded DNA isolated bySELEX (systemic evolution of ligands by exponential enrichment) usingMASP-2 or fragments thereof as selecting molecule. The leader sequenceof MASP-2 is shown elsewhere in this application.

[0138] MASP-2 activity may be controlled by the conversion of thepro-enzyme form of MASP-2 into activated MASP-2 through the action ofMASP-1 or any other substance simulating the activity of MASP-1.

EXAMPLES Example 1

[0139] Identification of MASP-2

[0140] Human plasma proteins and protein complexes, that bind tocarbohydrates in a calcium-dependent manner (i.e. lectins andlectin-associated proteins), were purified by affinity chromatography onmannan- and N-acetylglucosamine-derivatized Sepharose beads. PooledCPD-plasma (2.5 l), diluted with buffer containing EDTA and enzymeinhibitors were passed through Sepharose 2B CL and mannan-Sepharose. Athrombin inhibitor, PPACK (D-phenylalanyl-prolyl-arginyl-chloromethylketone) and CaCl₂were added. The pool was passed through Sepharose 2B-CLand mannan-Sepharose, and the proteins binding calcium-dependently tomannan-Sepharose were eluted with EDTA-containing buffer. The eluate wasrecalcified, passed through a GlcNAc-Sepharose column which was elutedas above to yield 20 ml “lectin preparation”.

[0141] This protein preparation was analyzed by SDS-PAGE and blottingonto a PVDF-membrane. Development of the blot with chicken antibodyraised against a bovine lectin preparation²⁵ revealed a protein with anM_(r) of 52 kDa as well as MBL at 32 kDa. The 52 kDa band was subjectedto NH₂-terminal amino acid sequence analysis. The sequence showedsimilarity to that of the previously described MASP (MASP1). Antibodyraised against a synthetic peptide representing the 19 NH₂-terminalamino acids (anti-N′ MASP-2 antiserum) recognized the 52 kDa molecule aswell as a molecule with a mobility corresponding to 20 kDa (FIG. 1, lane1). Under non-reducing conditions a polypeptide of 76 kDa was detectedusing the anti-N′-MASP-2 antiserum (FIG. 1, lane 2), indicating thepresence of intra-chain disulphide bonds. The 20 kDa polypeptide wasfound to have the same NH₂-terminal sequence as the 52 kDa polypeptideand is likely to represent a truncated form of the latter. The directlydetermined amino acid sequences (NH₂-terminal as well as those ofinternal peptides) are indicated in FIG. 6. Two dimensional SDS-PAGEwith the first dimension under nonreducing conditions and the seconddimension under reducing conditions showed the 52 kDa polypeptide to bepart of a disulphide-linked protein with an M_(r) of 76 kDa. Apolypeptide of 31 kDa (FIG. 1, lane 3), likely to represent theremaining part of the protein, was also recognized as part of the 76 kDaprotein by an antiserum (anti-C′ MASP-2) raised against syntheticpeptides representing sequences in the COOH-terminal part of the protein(determined by cDNA sequencing techniques; see below). The 76 kDa bandseen with the anti-N′ MASP-2 antibody under non-reducing conditions wasalso recognized by the anti-C′ MASP-2 antibody (FIG. 1, lane 4).

[0142]FIG. 1b depicts SDS-PAGE in two dimensions, the first dimensionunder non-reducing conditions. The lane was cut out, incubated in samplebuffer containing dithiothreitol (DTT), placed on top of anotherSDS-PAGE gel, and after electrophoresis, the gel was blotted and theblot developed with anti-N′ MASP-2 antibody. The positions of molecularweight markers are indicated.

Example 2

[0143] Preparation of Antibodies Against Mamman-binding LectinAssociated Serine Proteases

[0144] Animals, primed with BCG (Bacillus Calmette Guérin vaccine) wereimmunized with synthetic peptides coupled to PPD (tuberculin purifiedprotein derivative) according to C. Koch, The State Serum Institute,Copenhagen. Antibody designated anti-N′ MASP-1, anti-C′ MASP-1 andanti-N′ MASP-2 were from rabbits immunized with peptides correspondingto the first 19 amino acid residues of MASP-1, the last 19 amino acidresidues of MASP-1 and the first 19 amino acid residues of MASP-2,respectively. Chicken anti-C′ MASP-2 antibody was from chickensimmunized with a mixture of two peptides representing sequences in theC-terminal part of MASP-2 (residues 505 to 523 and 538 to 556). Allpeptides had an additional C-terminal cysteine for coupling. Antibodyand normal chicken IgG was purified from yolk²⁶. Monoclonal anti-MBLantibody, IgG₁-kappa (clone 131-1) and control IgG₁-kappa (clone MOPC21) were purified by Protein A affinity chromatography. F(ab′)₂ rabbitanti-human C4 and F(ab′)₂ rabbit anti-human Clq were produced by pepsindigestion of rabbit anti-human C4 and rabbit anti-human C1q (DAKO,Glostrup, Denmark). For staining of Western blots antibodies were usedat 1 μg/ml. Bound chicken antibody was visualized with rabbitanti-chicken IgG followed by peroxidase-labelled goat anti-rabbit IgGand development using the enhanced chemiluminescence technique. Boundmouse and rabbit antibodies were visualized with peroxidase-labelledrabbit anti-mouse IgG and peroxidase-labelled goat anti-rabbit IgG,respectively.

Example 3

[0145] Amino Acid Sequencing of the 52 kDa and the 20 kDa Polypeptides

[0146] The lectin preparation was concentrated, subjected to SDS-PAGE,and transferred to a PVDF membrane. Two strips were developed withanti-bovine lectin antibody²⁵. The rest of the blot was stained withCoomassie Brilliant Blue. The band corresponding to the immuno-stained52 kDa band, judged to represent about 5% of the total Coomassie-stainedproteins, was cut out and subjected to sequencing on an AppliedBiosystems protein sequencer. After production of anti-N′ MASP-2antibody, a similar Western blot was performed using the anti-N-MASP-2antibody. The NH₂-termini of the proteins in the 52 kDa and the 20 kDabands visualized with this antibody were sequenced. Peptides wereprepared by trypsin digestion of the proteins in the two bands fromanother blot, fractionated by reverse phase chromatography and thepeptides in the major peaks were subjected to sequencing.

Example 4

[0147] Cloning and Sequencing of MASP-2

[0148] The liver is the primary site of synthesis of C1r, C1s, andMASP-1. Thus, RNA from liver was used as template for RT-PCR withprimers deduced from the obtained peptide sequences. First strandsynthesis of cDNA was carried out with 1.3 μg human liver RNA using aFirst-Strand cDNA Synthesis Kit (Pharmacia). PCR was performed on thiscDNA using degenerate sense and antisense primers derived from the aminoacid sequences EYANDQER and KPFTGFEA, respectively. The PCR programconsisted of 1 cycle with annealing at 50° C.; 1 cycle with annealing at55° C., and 33 cycles with annealing at 60° C. The resulting 300 bp PCRproduct was cloned into the E. coli plasmid pCRII using the TA-cloningkit (InVitrogen) and the nucleotide sequence of the insert wasdetermined.

[0149] The nucleotide sequence of the resulting 300 bp RT-PCR productcontained an open reading frame (ORF) with a deduced amino acid sequenceconfirming the sequences of the peptides from which the primers werederived as well as that of another of the sequenced peptides. The insertof this plasmid was radioactivly labelled and used as a probe forscreening a total of 8×10⁵ clones in a commercial human liver library(Stratagene). Sixteen clones hybridized and the 4 longest (phl-1,2,3 and4) were completely sequenced. Sequence analysis revealed that all fourclones represent reverse transcripts of the same novel human mRNAspecies. The longest clone, phl-4, comprises 2475 bp starting with a 5′untranslated region of 36 bp followed by an ORF of 2061 bp and a 3′untranslated region of 378 bp ending with a poly-A tail. The nucleotidesequence of phl-4 is shown in FIG. 6 together with the translated aminoacid sequence. The sequences are deposited at the EMBL nucleotidesequence data base (accession no. Y09926). While the sequence of phl-1and -2 were in total agreement with phl-4, the nucleotide sequence ofphl-3 differs from phl-4 at two positions, a transversion at nucleotideposition 1147 (G to T) and a transition at position 1515 (C to T). Thefirst change leads to the replacement of Asp 356 with Tyr. Because allclones were isolated from a liver library transcribed from RNA isolatedfrom a single donor, the observed difference may represent apolymorphism in the MASP-2 gene, or is due to an error created duringconstruction of the library.

[0150] The amino acid sequences of the NH₂-terminus as well as allsequenced peptides were identified in the sequence deduced from clonephl-4. The ORF encodes a polypeptide chain of 686 amino acids includinga signal peptide of 15 residues. Omitting the signal peptide, thecalculated M_(r) is 74,153, in agreement with the 76 kDa observed onSDS-PAGE (FIG. 1), the isoelectric point is 5.43 and the molarextinction coefficient is 113,640 (i.e. OD_(280nm)=1.54 at 1 mg/ml). Incontrast to MASP-1 the sequence contains no sites for N-linkedglycosylation. The three amino acid residues which are essential for theactive centre in serine proteases (His 468, Asp 517, and Ser 618) arepresent.

Example 5

[0151] Comparison of MASP-2 to MASP-1, C1r and C1s.

[0152] The amino acid sequence deduced from the cDNA sequences ishomologous to those of MASP-1, C1r and C1s (FIG. 2). Notably, the domainorganization is common to these four proteins, featuring oneC1r/C1s-like domain, one epidermal growth factor-like (EGF-like) domain,followed by a second C1r/C1s-like domain, two complement control protein(CCP) domains, and a serine protease domain. The key residues involvedin the calcium-binding motif in the epidermal growth factor-like domainsare present in the obtained sequence, as well as in MASP-1, C1r and C1s.In addition, the substrate specificity related residue, 6 residuesbefore the active site serine, is aspartic acid in all four proteins.MASP-1, C1r, and C1s are all activated by cleavage of the peptide bondbetween the residues Arg and Ile located between the second CCP domainand the serine protease domain. The resulting polypeptide chains (thelargest referred to as the “heavy chain” and the smallest as “lightchain”) are held together by a disulphide bond. By analogy, our resultsindicate that the 52 kDa polypeptide, recognized by antibody against theN-terminal of MASP-2 after SDS-PAGE under reducing conditions, is theheavy chain of MASP-2, whereas the 31 kDa polypeptide, recognized byantibody against the C-terminal of MASP-2, is the light chain. As seenin FIG. 2, Arg and Ile are present in MASP-2 at the expected positionsbetween the second CCP domain and the protease domain.

[0153] Identities and similarities between the four proteins werestudied based on the alignment in FIG. 2. A bias of 6 was added to eachterm of the mutation data matrix (250PAMS) and a break penalty of 6 wasused. Identical residues in all four species are indicated by asterisks.The beginning of the C1r/C1s-like domains, the EGF-like domain and theCCP domains are indicated above the sequences. The aligned cysteines areshaded. The potential cleavage site between Arg and Ile residues, whichgenerates heavy and light chains, is identical to the site where theserine protease domain starts. The three amino acid residues, which areessential for the active centre in serine proteases (His 468, Asp 517and Ser 618), are indicated (⋄). The cysteines in the histidine-loop ofMASP-1 are marked (∇). The sequences obtained by amino acid sequencingof peptides are underlined. The identities between the proteins (FIG. 2)are all in the range of 39% to 45% and gives no clue to functionalrelatedness. The similarity, i.e. taking into account residues ofsimilar nature as well as identical residues, between the proteins (FIG.3b) are between 39 and 52% with the least similarity being betweenMASP-1 and C1s (39%) and the highest similarity between MASP-1 and C1r(52%) and between MASP-1 and MASP-2 (52%). MASP-2 shows similarity withC1r (46%) and C1s (47%). Whereas the relative identities gives no clueas to functional relatedness the similarity score between C1s and MASP-2is significantly higher than between C1s and MASP-1 while MASP-1 is moresimilar to C1r than to C1s, suggesting that MASP-2, like C1s, could be aC2 and C4 cleaving enzyme. Several features of the sequences suggestthat MASP-2, C1r and C1s have evolved by gene duplication and divergencefrom a MASP-1 ancestor. Only the MASP-1 sequence contains the histidineloop, characteristic of trypsin-like serine proteases²⁷. The active siteserine is encoded by a TCN codon (N is A, T, G or C) in MASP-1 as inmost serine proteases, whereas in MASP-2, C1r and C1s it is encoded byan AGY codon (where Y is T or C). In most serine proteases, includingMASP-1, a proline residue is found at the third position downstream fromthe active site serine, whereas a different amino acid is found inMASP-2, C1s and C1r (alanine in MASP-2 and C1s, valine in C1r). Based onthese analogies one may predict that the catalytic domain of MASP-2 isencoded by a single exon as in C1r and C1s, whereas most other serineproteases, including MASP-1²⁸, have split exons.

Example 6

[0154] MBL/MASP Complexes.

[0155] The lectin preparation described above was incubated inmicrotiter wells coated with monoclonal anti-MBL antibody, or, as anegative control, wells coated with non-specific monoclonalimmunoglobulin of the same subclass. The proteins captured by theantibody were eluted and analyzed by SDS-PAGE/Western blotting. Theresults (FIG. 3a) show that the anti-MBL antibody, in addition tobinding MBL, captures both MASP-1 and MASP-2. Microtiter wells werecoated with monoclonal anti-MBL or control monoclonal murine IgG1,incubated with either one of two different lectin preparations (a andb), and the bound proteins were eluted and analysed by SDS-PAGE underreducing conditions and Western blotting. Blot a was developed withanti-MBL antibody, blot b with anti-C′ MASP-1 antibody and blot c withanti-N′ MASP-2 antibody. Lane 1 represents unfractionated lectinpreparation a. Lanes 3 and 4 represent eluates from wells coated withanti-MBL antibody and incubated with lectin preparation b and a,respectively, while lanes 2 and 5 represent eluates from wells coatedwith normal IgG and incubated with lectin preparation b and a,respectively.

[0156] Fractions from gel permeation chromatography (GPC) of the lectinpreparation on Superose 6B CL were analyzed for MBL, MASP-1 and MASP-2(FIG. 3a). The lectin preparation was subjected to GPC on a Superose 6column in buffer containing calcium. MBL was eluted in a main peak at avolume (V_(e)) corresponding to an M_(r) of 750 kDa, and a smaller peakat a position corresponding to 350 kDa. Panel A shows the results ofanalysis of the fractions by Western blotting using monoclonal anti-MBLantibody. The band at about 60 kDa is seen in all MBL preparations andis recognized by all the anti-MBL antibodies tested (monoclonal as wellas polyclonal) and thus probably represents a non-reducible dimer of the32 kDa polypeptide chain. Panel B shows the same analysis using anti-N′MASP-2 antibody (developing the upper band of 52 kDa) followed byanti-C′ MASP-1 antibody (developing the lower band of 31 kDa). Forpurely technical reasons the 20 kDa truncated MASP-2 is not seen in thispicture where the blot was partially stripped between the incubationswith anti-MASP-2 and anti-MASP-1. The arrows on the chromatogramindicate the void volume (1) and the elution positions for the followingmarker proteins IgM (2), C1q (3), thyroglobulin (4), IgG (5) and serumalbumin (6).

[0157] Masp-1 and MASP-2 coelute largely with the high molecular weightMBL. Chromatography of the MBL preparation at pH 5 revealed that noMASP-1 or MASP-2 was associated with MBL. See, Tan et al. (1996, BiochemJ. 319: 329-332).

Example 7

[0158] Complement Activation.

[0159] The classical complement activation pathway, as well as theMBL-initiated pathway involves the generation of a C3 convertingcomplex, C4b2b, through enzymatic activation of C4 and C2. In the C1complex (C1qr₂s₂) this specific protease activity is exhibited by C1safter activation of the enzyme by C1r. Upon activation of C4, a reactivethiol ester is exposed and C4b covalently binds to nearby amino orhydroxyl groups.

[0160] The C4 activating potentials of MASP-1 and MASP-2, and C1r andC1s, were compared. This was accomplished by separating a C1 preparationand an MBL/MASP preparation by SDS-PAGE followed by Western blotting.The blot was examined for C4 converting activity by incubation withhuman serum at 37° C., followed by detection of deposited C4b usinganti-C4 antibodies.

[0161] C1 was purified by incubating IgG-coupled Sepharose beads withhuman serum at 4° C. The beads were washed and incubated at 37° C. for30 minutes for activation of C1r and C1s. The beads were suspended innon-reducing sample buffer and, without boiling, subjected to SDS-PAGE,followed by blotting in the absence of SDS. A similar blot was made ofan MBL preparation produced in the absence of enzyme inhibitors (TheState Serum Institute, Copenhagen). Strips of the blots were incubatedfor 30 minutes at 37° C. with 1.1% (v/v) human MBL-deficient serum,depleted of C1q by fractionation on Biorex 70. The blots were developedwith biotinylated F(ab′)₂ anti-C4 antibody followed byperoxidase-labelled streptavidin and luminescence reagent. Parallelblots were treated with a serine protease inhibitor(aminoethylbenzenesulfonyl fluoride), which was also present duringincubation with serum. Other strips were directly developed withantibodies.

[0162] The results in FIG. 4 show that C4 was deposited at a positioncorresponding to the MASP-2 band, whereas no C4 deposition was observedat positions corresponding to MASP-1. MASP-1 was present in theactivated state as shown by SDS-PAGE under reducing conditions where itappears as two bands at about 30 kDa and 70 kDa, respectively (notshown). The observed C4 activation and deposition was inhibited byserine protease inhibitors (FIG. 4). It was also observed that no C4activating activity could be detected when MBL/MASP was prepared in thepresence of enzyme inhibitors added throughout the purificationprocedure. A preparation of C1 was analyzed similarly and C4 deposition,which could be inhibited by enzyme inhibitors, was observed at aposition corresponding to C1r and C1s, which are not separated by thetechnique employed.

[0163]FIG. 4 is a representation of Western blots demonstrating theactivation of C4 by C1s and MASP-2. Panel A shows a Western blot of C1separated under non-reducing conditions, and without heating the samplebefore electrophoresis. Lane 1 was developed with anti-C1s antibody.Lane 2 was incubated with human serum followed by anti-C4 antibody. Lane3 was as lane 2 except for the presence of serine protease inhibitorsduring the incubation with serum. Panel B shows a Western blot of an MBLpreparation separated as in A. Lane 1 was developed with anti-N′ MASP-1,lane 2 with anti-N′ MASP-2. Lane 3 was incubated with human serum at 37°C. followed by anti-C4. In lane 4 the blot was preincubated with serineprotease inhibitors and the incubation with serum was also in thepresence of inhibitors. MASP-1 shows a higher M_(r), than MASP-2 due toglycosylation¹⁷ and a polypeptide chain 9 amino acids longer.

[0164] Our results emphasize the similarity between complementactivation through the MBLectin pathway of the innate immune system andthe classical pathway of complement activation (FIG. 5). Activation viathe classical pathway is associated with the specific immune responsefound only in vertebrates, while the MBLectin pathway and thealternative pathway rely on innate recognition of foreign organisms andare thus likely to predate the evolution of the specific immune system.All pathways converge on the activation of the central component C3 intoC3b, which binds covalently to the microbial surface and mediates theeffector functions of complement.

[0165] In both the classical and MBLectin pathways, the initiatingmolecular complexes are composed of an oligomeric ligand-bindingcomponent (MBL or C1q, respectively) which, on reacting with ligands,activates the two associated serine proteases (MASP-1 and MASP-2 or C1rand C1s, respectively).

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[0199] 34) Mackinnon, C. M., Carter, P. E., Smyth, S. J., Dunbar, B. &Fothergill, J. E. Molecular cloning of cDNA for human complementcomponent C1s. The complete amino acid sequence. Eur. J. Biochem. 169,547-553 (1987).

[0200] 35) Tosi, M., Duponchel, C., Meo, T. & Julier, C. Complete cDNAsequence of human complement C1s and close physical linkage of thehomologous genes C1s and C1r. Biochemistry 26, 8516-8524 (1987).

What is claimed is:
 1. A substantially pure mannin-binding lectinassociated serine protease-2 (MASP-2) polypeptide.
 2. The polypeptide ofclaim 1, said polypeptide being capable of associating withmannan-binding lectin (MBL).
 3. The polypeptide of claim 1, saidpolypeptide being conjugated to a label or toxin.
 4. A polypeptidecontaining the sequence identified as SEQ ID NO.
 1. 5. A polypeptideaccording to claim 4 with a molecular mass of 20K.
 6. A polypeptide witha molecular mass of 52K and containing the sequence identified as SEQ IDNO
 1. 7. The polypeptide of claim 1, said polypeptide having serineprotease activity.
 8. A polypeptide of claim 1, said polypeptide beingcapable of MASP-2 activity in an in vitro assay for MBLectin complementpathway function.
 9. A polypeptide according to claim 1, saidpolypeptide being capable of competitively inhibiting MASP-2 serineprotease activity.
 10. A polypeptide according to claim 1 comprising afragment of the polypeptide of SEQ ID NO:2, said polypeptide being acompetitive inhibitor of complexing of MEL/MASP-2.
 11. A polypeptideaccording to claim 5 or claim 6, said polypeptide having the amino acidsequence of SEQ ID NO:2.
 12. A compound capable of competitivelyinhibiting serine protease activity of MASP-2 or a fragment thereof. 13.An isolated nucleic acid molecule of claim, the molecule comprising anucleotide sequence encoding a polypeptide having sequence that is atleast 85% identical to the sequence of SEQ ID NO:1 or
 2. 14. An isolatednucleic acid sequence encoding a mannan-binding lectin associated serineprotease-2 (MASP-2) polypeptide according to claim
 1. 15. A nucleic acidvector comprising the nucleic acid molecule of claim
 14. 16. The nucleicacid vector of claim 15 wherein said vector is an expression vector. 17.The vector of claim 16, further comprising a regulatory element.
 18. Anantibody produced by administering an MASP-2 polypeptide according toclaim 1 to an antibody producing animal.
 19. An antibody thatselectively binds to MASP-2.
 20. The antibody of claim 18 or claim 19,wherein said antibody is a monoclonal antibody.
 21. The antibody ofclaim 18 or 19, said antibody being coupled to a compound comprising adetectable marker.
 22. A pharmaceutical composition comprising thepolypeptide of claim 1 or the antibody of claims 18 or
 19. 23. A methodfor detecting mannin-binding lectin associated serine protease-2(MASP-2), said method comprising: (a) obtaining a biological sample; (b)contacting said biological sample with a MASP-2 polypeptide specificbinding partner that specifically binds MASP-2; and (c) detecting saidcomplexes, if any, as an indication of the presence of mannin-bindinglectin associated serine protease-2 in said sample.
 24. A methodaccording to claim 23, in which the specific binding partner is anantibody.
 25. A method for detecting MASP-2, said method comprising anassay for MASP-2 complement fixing activity.
 26. The methods of claim 23or 24 for quantitative assay of MASP-2 or MASP-2 activity in biologicalsamples.
 27. A method for detecting MASP-2 nucleic acid expression,comprising detecting RNA having a sequence encoding a MASP-2 polypeptideby mixing the sample with a nucleic acid probe that specificallyhybridizes under stringent conditions to the nucleic acid of claim 13 or14.
 28. A method for treating patients deficient in MASP-2 byadministering to the patient the peptide of claim
 1. 29. A method fortreating patients deficient in MASP-2 by administering to the patientnucleic acid according to claim 13 or
 14. 30. A method for inhibitingthe activity of MASP-2 by administering to the subject a compound thatinhibits expression or activity of MASP-2.
 31. The method of claim 27 inwhich the compound is a MASP-2 anti-sense nucleic acid sequence.
 32. Themethod of claim 30 comprising administering a compound that inhibitscomplexing of MBL and MASP-2.
 33. An assay for polymorphisms in thenucleic acid sequence encoding MASP-2.
 34. A method of detecting thepresence of MASP-2-encoding nucleic acid in a sample, comprising mixingthe sample with at least one nucleic acid probe capable of forming acomplex with MASP-2-encoding nucleic acid under stringent conditions,and determining whether the probe is bound to sample nucleic acid.
 35. Anucleic acid probe capable of forming a complex with MASP-2-encodingnucleic acid under stringent conditions.
 36. An assay for polymorphismsin the polypeptide sequence comprising MASP-2 or its precursor.
 37. Amethod for diagnosing a disorder associated with aberrant expression ofMASP-2, comprising obtaining a biological sample from a patient andmeasuring MASP-2 expression in said biological sample, wherein increasedor decreased MASP-2 expression in said biological sample compared to acontrol indicates that said patient suffers from a disorder associatedwith aberrant expression of MASP-2.
 38. A method for diagnosing adisorder associated with aberrant activity of MASP-2, comprisingobtaining a biological sample from a patient and measuring MASP-2activity in said biological sample, wherein increased or decreasedMASP-2 activity in said biological sample compared to a controlindicates that said patient suffers from a disorder associated withaberrant activity of MASP-2.
 39. A method of assaying for activityMBL-complexed MASP, the method comprising providing a sample to beassayed and substantially reducing any artifact resulting fromactivation of the classical complement fixing pathway by conducting theassay in the presence of an ionic strength high enough to effectivelyreduce activation of the classical complement fixing pathway but not sohigh as to substantially interfere with activity of MBL-complexed MASP.